Factor VIII (FVIII) is the protein deficient or functionally defective in hemophilia A, an X chromosome-linked bleeding disorder affecting 1/5,000 males. To avoid significant morbidity and mortality, patients are treated by protein replacement with recombinant-derived FVIII produced in mammalian cells. Although recombinant FVIII (rFVIII) has significantly reduced the risk of virus infection, treatment is still problematic due to the high cost to manufacture rFVIII, shortage of supply, limited access to peripheral veins, and development of inhibitory antibodies. Our previous studies demonstrated that FVIII production in mammalian cells is limited due to inefficient folding and transport through the early secretory pathway. The long-term goal of the proposed research is to elucidate key mechanisms that limit FVIII secretion in order to increase the production of rFVIII for treatment of hemophilia A. We will test the following three hypotheses (*): * FVIII secretion is limited by protein aggregation, protein chaperone interactions, and oxidative stress. Specific Aim 1: To define the chaperone interactions which regulate FVIII aggregation, folding, and trafficking. *FVIII synthesis activates an adaptive cellular response that facilitates FVIII folding and secretion. Specific Aim 2: To unravel the molecular mechanism(s) by which IRE11/XBP1 and ATF61 promote proper FVIII folding and trafficking within the early secretory pathway. * Productive FVIII folding and trafficking requires interaction with the cargo receptor LMAN1/MCFD2, through asparagine-linked glycans within the B domain. Specific Aim 3: To elucidate the requirement for LMAN1/MCFD2 in FVIII secretion. The approach will use a combination of biochemical, cell biological, and novel genetic mouse models to dissect fundamental processes that direct and limit FVIII trafficking through the secretory pathway. The information will be vital to the future development of improved gene therapy protocols for hemophilia A. The findings will provide fundamental mechanistic insight into the processes that regulate protein folding and trafficking within the secretory pathway. These studies will have much broader impact toward understanding the etiology of many genetic and acquired diseases of protein misfolding and the discoveries may uncover novel therapeutic avenues for multiple disease states. PUBLIC HEALTH RELEVANCE: FVIII is the protein deficient in hemophilia A. Although protein replacement with recombinant FVIII is the preferred treatment, it is very costly. As FVIII production is limited due to protein misfolding, our studies to elucidate mechanisms that improve FVIII folding that should increase production of FVIII. The findings will impact all cell/gene therapy approaches to treat hemophilia A.